CN101671659A - Pectase and preparation method thereof - Google Patents
Pectase and preparation method thereof Download PDFInfo
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- CN101671659A CN101671659A CN200910019143A CN200910019143A CN101671659A CN 101671659 A CN101671659 A CN 101671659A CN 200910019143 A CN200910019143 A CN 200910019143A CN 200910019143 A CN200910019143 A CN 200910019143A CN 101671659 A CN101671659 A CN 101671659A
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- aspergillus niger
- polygalacturonase
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Abstract
The invention relates to pectase and a preparation method thereof. The pectase belongs to the pectase from a microbial source, and is characterized in that: a. enzymology characteristics: the optimalpH value is 3.5, and the stable pH value is 2.5-6.0; the optimal operative temperature is 50 DEG C, and the temperature stability range is 30-55 DEG C; b. aspergillus niger strains are selected and preserved as zymogenic strains in CICC with the preservation NO. 2238, wheat bran is used as a main raw material; and the pectase is prepared through the processes of solid fermentation, soaking extraction, filtration enrichment, liquation purification, ultrafiltration and fine filtration, wherein the fermentation enzyme-producing wet mold culture level is more than and equal to 41,000u/g; and carrying out soaking extraction, ultrafiltration and fine filtration, wherein the recovery rate of liquid enzymes is more than and equal to 85 percent and the recovery rate of solid enzymes is more than and equal to 80 percent, which conform the food hygienic standard. The invention provides the pectase with high enzyme activity and good stability and the preparation method thereof using the wheat branas the main raw material and having high fermentation level, high extraction and purification yield, high equipment utilization ratio and low production cost. The invention is suitable for industriesof fruit processing, wine production, bast fiber degumming, wood preservation, biological pulping, environmental protection, dirt softening and feed and the like.
Description
Technical field
The present invention is a kind of polygalacturonase and preparation method thereof.Belong to the polygalacturonase from microbial source, particularly being preserved in CICC numbering 2238 with aspergillus niger (Aspergillus niger) is the zymogenic bacteria kind, adopts polygalacturonase of solid fermentation process preparation and preparation method thereof.
Background technology
Polygalacturonase is the general name that acts on the class of enzymes of pectin substance, and major function is by the glycosidic link in cracking or the β cancellation effect cut-out pectin substance, makes pectin substance be cracked into polygalacturonic acid.
In recent years; the biological degradation of pectin substance causes the extensive concern of Chinese scholars day by day; the biology that polygalacturonase is used for pectin substance falls Application Areas and constantly expands; not only be used for foodstuffs industry such as aspects such as processing fruits and Production of Wine; be widely used in also that crudefiber crop is come unstuck, wood preservation, bio-pulping, environment protection, dirt softening handle and industries such as feed in; need with nearly ten thousand tons of polygalacturonases according to market analysis China is annual, the polygalacturonase product can not satisfy the demand in market far away.
In the prior art, be carbon source, consume a large amount of valuable grain resources with corn, soybean, solid fermentation is produced polygalacturonase, and the enzymatic production level is low, only about 15000u/g, enzyme extraction, purification yield are low, cause fermentation unit throughput low, polygalacturonase production cost height.
Heat in the invention
The objective of the invention is to avoid above weak point of the prior art, and the polygalacturonase of a kind of enzyme activity height, good stability is provided.
The present invention also aims to provide a kind of is main raw material with wheat bran, enzymatic production level height, extraction purification yield height, plant factor height, the polygalacturonase preparation method that production cost is low.
Purpose of the present invention can reach by following measure:
Polygalacturonase of the present invention is characterized in that:
A. enzymatic property
Optimal pH 3.5 is stablized pH2.5-6.0; Optimum temperature is 50 ℃, and the temperature stability scope is 30 ℃-55 ℃;
B. select aspergillus niger strain (Aspergillus niger) for use, be preserved in CICC numbering 2238 and be the zymogenic bacteria kind, wheat bran is main raw material, adopts solid fermentation, immersion extraction, filtering and concentrating, dissolved purifying, ultrafiltration, the preparation of smart filtering technology, reaches following technical indicator:
1. the wet Qushui River of enzymatic production is flat: 〉=41000u/g;
2. soak extraction, ultrafiltration, smart filter, the liquid enzymes rate of recovery 〉=85%, the solid enzyme rate of recovery 〉=80% reaches the food grade hygienic standard.
It is that the very high good quality strain of enzyme is produced in a strain that the aspergillus niger strain that the present inventor selects for use (Aspergillus niger) is preserved in CICC numbering 2238, and the polygalacturonase of output has enzyme activity height, good stability characteristic.
The present inventor finds unexpectedly, by the cereal shell or not hulled grain participate in significantly improving as the enzymatic productivity that the solid fermentation main culture based raw material can make aspergillus niger (Aspergillus niger) be preserved in CICC numbering 2238.Wheat bran is as the by-product of whole meal flour processing industry, and the source is abundant.To select wheat bran for use be that main carbon source had both been saved valuable grain resource, improved the enzymatic productivity that aspergillus niger (Aspergillus niger) is preserved in CICC numbering 2238 again.For productive rate and its production cost of reduction of improving polygalacturonase of the present invention are made great contributions to
Adopt the solid fermentation process method, improved the utilization ratio that feeds intake of fermentation equipment greatly, thereby improved the throughput of equipment.Soak the enforcement of extraction, ultrafiltration and concentration, dissolved purifying, ultrafiltration, smart filter, vacuum drying process, for the raising of enzyme extraction purification yield provides technical support.
Purpose of the present invention can also reach by following measure:
Polygalacturonase of the present invention, it is characterized in that the aspergillus niger described in the b (Aspergillus niger) is preserved in CICC and numbers 2238 zymogenic bacteria kinds with cobalt 60 rays, the processing of excess ultraviolet mutagenesis, give good characteristics such as its stabilization characteristics of genetics is good, product enzyme speed is fast, product enzyme level height.
Polygalacturonase of the present invention is characterized in that moisture content in the solid fermentation raw material between 40%-50%, is an optimized technical scheme disclosed by the invention.
A kind of preparation method of polygalacturonase of the present invention is provided below, it is characterized in that comprising the steps:
A. liquid spawn is cultivated in advance: substratum 10-25 weight part, add water to 100 weight parts, and regulate pH 5-6, sterilized 30-35 minute for 121 ℃, be cooled to 30 ℃-35 ℃, access (Aspergillus niger) is preserved in CICC and numbers 2238 bacterial classifications, gets the pre-nutrient solution of liquid spawn; 30 ℃-40 ℃ of controlled temperature, ventilation 45-60m
3/ h, pressure 0.05Mpa-0.08Mpa cultivated 28-32 hour, got liquid spawn, was equipped with solid state fermentation and used;
B. solid state fermentation: with the cereal shell is 120 ℃-123 ℃ of main fermentation raw materials boiling 50-60 minute, is cooled to 30 ℃-35 ℃, inserts the liquid spawn of step a preparation, stir evenly, make material initial water content 40%-50%, ventilation: 50-60m3/h, culture cycle 5.0 days; Fermentation level 〉=41000u/g;
C. adopt and soak extraction, filtering and concentrating, dissolved purifying, ultrafiltration, smart filter, adjusting pH to 4.2 ± 0.1, liquid enzymes yield 〉=86% reaches the food grade hygienic standard.
The application of polygalacturonase of the present invention, it is characterized in that being applicable to that processing fruits, Production of Wine, crudefiber crop are come unstuck, wood preservation, bio-pulping, environment protection, dirt softening handle and industries such as feed in.
Polygalacturonase of the present invention and preparation method thereof has outstanding substantive distinguishing features and obvious improvement compared to existing technology, can produce following positively effect:
1. the polygalacturonase of a kind of enzyme activity height, good stability is provided.
2. it is that the very high good quality strain of enzyme is produced in a strain that the aspergillus niger strain of selecting for use (Aspergillus niger) is preserved in CICC numbering 2238, and the polygalacturonase of output has enzyme activity height, good stability characteristic.
3. be main raw material with wheat bran, both saved valuable grain resource, further improved the product enzyme level that aspergillus niger strain (Aspergillus niger) is preserved in CICC numbering 2238 again, enzymatic production level 〉=41000u/g
4. optimize the solid-state fermentation process condition, the enforcement of soaking extraction, filtering and concentrating, dissolved purifying, ultrafiltration, smart filtering technology makes and extracts the purification yield height, the plant factor height, and the polygalacturonase production cost reduces greatly.
Embodiment
The present invention does further ocean below in conjunction with embodiment and states:
Embodiment 1 solid fermentation prepares polygalacturonase of the present invention
A. liquid spawn is cultivated in advance
With 100 kilograms in dextrin, 10 kilograms of peptones, 20 kilograms of corn steep liquors, soybean cake powder drops in the 1000L reactor for 12 kilograms, adds water to 1000 kilograms of total charging capacitys, and regulates pH to 6.0; Be warming up to 121 ℃, insulated sterilizing 35 minutes.Be cooled to 30 ℃, insert aspergillus niger strain (Aspergillus niger) and be preserved in CICC numbering 2238, get the pre-nutrient solution of liquid spawn; 30 ℃ of controlled temperature, ventilation 60m
3/ h, jar internal pressure 0.05Mpa-0.08Mpa, culture cycle 32 hours gets liquid spawn, is equipped with solid fermentation and uses;
B. solid state fermentation
3000 kilograms in wheat bran, under 121 ℃ of temperature, boiling 60 minutes; Be cooled to 35 ℃; Stir the diluted liquid spawn of inserting step a preparation down, stir, the initial water content that makes fermentation raw material is 45%; At 35 ℃ of temperature, ventilation 60m
3/ h, fermentation culture 5.0 days, fermentation level: 〉=41000u/gl;
C. adopt and soak extraction, filtering and concentrating, dissolved purifying, ultrafiltration, smart filter, adjusting pH to 4.2 ± 0.1, the allotment can, liquid enzymes yield 〉=86% reaches the food grade hygienic standard.
Embodiment 2 solid fermentations prepare polygalacturonase of the present invention
A. liquid spawn is cultivated in advance
With 100 kilograms of starch, 10 kilograms of peptones, 20 kilograms of corn steep liquors, alpha-amylase 100ml, calcium chloride drops in the 1000L reactor for 5 kilograms, adds water to 1000 kilograms of total charging capacitys, and regulates pH to 6.0; Be warming up to 121 ℃, insulated sterilizing 35 minutes.Be cooled to 30 ℃, insert aspergillus niger strain (Aspergillus niger) and be preserved in CICC numbering 2238, get the pre-nutrient solution of liquid spawn; 34 ℃ of controlled temperature, ventilation 60m
3/ h, jar internal pressure 0.05Mpa-0.08Mpa, culture cycle 30 hours gets liquid spawn, is equipped with solid fermentation and uses;
B. solid state fermentation
3000 kilograms in wheat bran, under 123 ℃ of temperature, boiling 50 minutes; Be cooled to 35 ℃; Stir the diluted liquid spawn of inserting step a preparation down, stir, the initial water content that makes fermentation raw material is 50%; At 35 ℃ of temperature, ventilation 60m
3/ h, fermentation culture 5.0 days, fermentation level: 〉=41000u/gl;
C. adopt and soak extraction, filtering and concentrating, dissolved purifying, ultrafiltration, smart filter, adjusting pH to 4.0 ± 0.1, the allotment can, liquid enzymes yield 〉=88% reaches the food grade hygienic standard.
Embodiment 3 solid fermentations prepare polygalacturonase of the present invention
A. liquid spawn is cultivated in advance
With 100 kilograms of malt syrups, 20 kilograms of corn steep liquors, 12 kilograms of soybean cake powder drop in the 1000L reactor, add water to 1000 kilograms of total charging capacitys, and regulate pH to 5.6; Be warming up to 121 ℃, insulated sterilizing 35 minutes.Be cooled to 35 ℃, insert aspergillus niger strain (Aspergillus niger) and be preserved in CICC numbering 2238, get the pre-nutrient solution of liquid spawn; 34 ℃ of controlled temperature, ventilation 60m
3/ h, jar internal pressure 0.05Mpa-0.08Mpa, culture cycle 28 hours gets liquid spawn, is equipped with solid fermentation and uses;
B. solid state fermentation
3000 kilograms in wheat bran, under 123 ℃ of temperature, boiling 50 minutes; Be cooled to 35 ℃; Stir the diluted liquid spawn of inserting step a preparation down, stir, the initial water content that makes fermentation raw material is 53%; At 35 ℃ of temperature, ventilation 50m
3/ h, fermentation culture 5.0 days, fermentation level: 〉=41000u/gl;
C. adopt and soak extraction, filtering and concentrating, dissolved purifying, ultrafiltration, smart filter, adjusting pH to 4.5 ± 0.1, the allotment can, liquid enzymes yield 〉=88% reaches the food grade hygienic standard.
Claims (5)
1. polygalacturonase is characterized in that:
A. enzymatic property
Optimal pH 3.5 is stablized pH2.5-6.0; Optimum temperature is 50 ℃, and the temperature stability scope is 30 ℃-55 ℃;
B. select aspergillus niger strain (Aspergillus niger) for use, be preserved in CICC numbering 2238 and be the zymogenic bacteria kind, wheat bran is main raw material, adopts solid fermentation, immersion extraction, filtering and concentrating, dissolved purifying, ultrafiltration, the preparation of smart filtering technology, reaches following technical indicator:
1. the wet Qushui River of enzymatic production is flat: 〉=41000u/g;
2. soak extraction, ultrafiltration, smart filter, the liquid enzymes rate of recovery 〉=85%, the solid enzyme rate of recovery 〉=80% meets the food grade hygienic standard.
2. according to the polygalacturonase of claim 1, it is characterized in that the aspergillus niger described in the b (Aspergillus niger) is preserved in CICC and numbers 2238 zymogenic bacteria kinds with cobalt 60 rays, the processing of excess ultraviolet mutagenesis, can give good characteristics such as its stabilization characteristics of genetics is good, product enzyme speed is fast, product enzyme level height.
3. according to the polygalacturonase of claim 1, it is characterized in that moisture content in the solid fermentation raw material is between 40%-53%.
4. the preparation method of the aspartic protease of a claim 1 is characterized in that comprising the steps:
A. liquid spawn is cultivated in advance: substratum 10-25 weight part, add water to 100 weight parts, and regulate pH5-6, sterilized 30-35 minute for 121 ℃, be cooled to 30 ℃-35 ℃, access (Aspergillus niger) is preserved in CICC and numbers 2238 bacterial classifications, gets the pre-nutrient solution of liquid spawn; 30 ℃-40 ℃ of controlled temperature, ventilation 45-60m
3/ h, pressure 0.05Mpa-0.08Mpa cultivated 28-32 hour, got liquid spawn, was equipped with solid state fermentation and used;
B. solid state fermentation: with the cereal shell is 120 ℃-123 ℃ of main fermentation raw materials boiling 50-60 minute, is cooled to 30 ℃-35 ℃, inserts the liquid spawn of step a preparation, stirs evenly, and makes material initial water content 40%-50%, ventilation: 50-60m
3/ h, culture cycle 5.0 days; Fermentation level 〉=41000u/g;
C. adopt and soak extraction, filtering and concentrating, dissolved purifying, ultrafiltration, smart filter, adjusting pH to 4.0 ± 0.1-4.5 ± 0.1, liquid enzymes yield 〉=86% reaches the food grade hygienic standard.
5. the application of the polygalacturonase of claim 1 is characterized in that being applicable to that processing fruits, Production of Wine, crudefiber crop are come unstuck, wood preservation, bio-pulping, environment protection, dirt are softening handles and industries such as feed are used.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200910019143A CN101671659A (en) | 2009-09-25 | 2009-09-25 | Pectase and preparation method thereof |
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| CN200910019143A CN101671659A (en) | 2009-09-25 | 2009-09-25 | Pectase and preparation method thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101857857A (en) * | 2010-05-18 | 2010-10-13 | 青岛康地恩生物科技有限公司 | Method for preparing food-grade acid pectase preparation |
| CN101948820A (en) * | 2009-08-27 | 2011-01-19 | 山东隆科特酶制剂有限公司 | Acidic proteinase and preparation method thereof |
| CN102342567A (en) * | 2011-06-20 | 2012-02-08 | 海南大学 | Pepper peeling method |
-
2009
- 2009-09-25 CN CN200910019143A patent/CN101671659A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101948820A (en) * | 2009-08-27 | 2011-01-19 | 山东隆科特酶制剂有限公司 | Acidic proteinase and preparation method thereof |
| CN101857857A (en) * | 2010-05-18 | 2010-10-13 | 青岛康地恩生物科技有限公司 | Method for preparing food-grade acid pectase preparation |
| CN102342567A (en) * | 2011-06-20 | 2012-02-08 | 海南大学 | Pepper peeling method |
| CN102342567B (en) * | 2011-06-20 | 2014-04-02 | 海南大学 | Pepper peeling method |
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Application publication date: 20100317 |